Since the discovery of Shigella dysenteriae type 1 (Shiga's bacillus) more than a century ago (R. Shields and W. Burnett, Zentl. Bakterio., 1898, 24, 817-828), shigellosis or bacillary dysentery has been known as a serious infectious disease, occurring in humans only (T. G. Keusch and M. L. Bennish, Shigellosis, Plenum Medical Book Company, New York, 1991, p. 593-620). In a recent survey of the literature published between 1966 and 1997 (K. L. Kotloff, J. P. Winickoff, B. Ivanoff, J. D. Clemens, D. L. Swerdlow, P. J. Sansonetti, G. K. Adak and M. M. Levine, Bull. WHO, 1999, 77, 651-666), the number of episodes of shigellosis occurring annually throughout the world was estimated to be 164.7 million, of which 163.2 million were in developing countries. Up to 1.1 million annual deaths were associated with shigellosis during the same period. Occurrence of the disease is seen as a correlate of sanitary conditions, and those are not likely to improve rapidly in areas at risk.
The financial status of the populations in which shigellosis exists in its endemic forms, as well as the emerging resistance to antimicrobial drugs (M. U. Khan, Int. J. Epidemiol., 1985, 14, 607-613; B. A. Iwalokun, G. O. Gbenle, S. I. Smith, A. Ogunledun, K. A. Akinsinde and E. A. Omonigbehin, J. Health Popul. Nutr., 2001, 19, 183-190), limit the impact of the latter. Of the four species of Shigellae, S. flexneri is the major responsible for the endemic form of the disease, with serotype 2a being the most prevalent. The critical importance of the development of a vaccine against Shigellae infections was first outlined in 1987 (World Health and Organization, Bull. W.H.O., 1987, 65, 17-25). Due to increasing resistance of all groups of Shigellae to antibiotics (S. Ashkenazi, M. May-Zahav, J. Sulkes and Z. Sanwa, Antimicrob. Agents Chernother., 1995, 39, 819-823) vaccination remained a high priority as stated by the World Health Organization ten years later (WHO, Weekly Epidemiol. Rec., 1997, 72, 73-79). In the meantime, several experimental vaccines have gone through field evaluation (T. S. Coster, C. W. Hoge, L. L. van der Verg, A. B. Hartman, E. V. Oaks, M. M. Venkatesan, D. Cohen, G. Robin, A. Fontaine-Thompson, P. J. Sansonetti and T. L. Hale, Infect. Immun., 1999, 67, 3437-3443; J. H. Passwell, E. Harlev, S. Ashkenazi, C. Chu, D. Miron, R. Ramon, N. Farzan, J. Shiloach, D. A. Bryla, F. Majadly, R. Roberson, J. B. Robbins and R. Schneerson, Infect. Immun., 2001, 69, 1351-1357) but there are as yet no licensed vaccines for shigellosis.
Shigella's lipopolysaccharide (LPS) is a major surface antigen of the bacterium. The corresponding O—SP domain (O—SP) is both an essential virulence factor and the target of the infected host's protective immune response (D. Cohen, M. S. Green, C. Block, T. Rouach and I. Ofek, J. Infect. Dis., 1988, 157, 1068-1071; D. Cohen, M. S. Green, C. Block, R. Slepon and I. Ofek, J. Clin. Microbiol., 1991, 29, 386-389). Indeed, using the pulmonary murine model for shigellosis, it was demonstrated that the presence locally, preliminary to infection, of a secretory antibody of isotype A specific for an epitope located on the O—SP moiety of the LPS of S. flexneri 5a, prevented any host homologous infection (A. Phalipon, M. Kauffmann, P. Michetti, J.-M. Cavaillon, M. Huerre, P. Sansonetti and J.-P. Krahenbuhl, J. Exp. Med., 1995, 182, 769-778). Based on the former hypothesis that serum IgG anti-LPS antibodies may confer specific protection against shigellosis (J. B. Robbins, C. Chu and R. Schneerson, Clin. Infect. Dis., 1992, 15, 346-361), several polysaccharide-protein conjugates, targeting either Shigella sonnei, S. dysenteriae 1 or S. flexneri serotype 2a, were evaluated in humans (J. H. Passwelle, E. Harlev, S. Ashkenazi, C. Chu, D. Miron, R. Ramon, N. Farzan, J. Shiloach, D. A. Bryla, F. Majadly, R. Roberson, J. B. Robbins and R. Schneerson, Infect. Immun., 2001, 69, 1351-1357; D. N. Taylor, A. C. Trofa, J. Sadoff, C. Chu, D. Bryla, J. Shiloach, D. Cohen, S. Ashkenazi, Y. Lerman, W. Egan, R. Schneerson and J. B. Robbins, Infect. Immun., 1993, 61, 3678-3687). In the case of S. sonnei, recent field trials allowed Robbins and co-workers to demonstrate the efficacy of a vaccine made of the corresponding detoxified LPS covalently linked to recombinant exoprotein A (D. Cohen, S, Ashkenazi, M. S. Green, M. Gdalevich, G. Robin, R. Slepon, M. Yavzori, N. Orr, C. Block, I. Ashkenazi, J. Sherner, D. N. Taylor, T. L. Hale, J. C. Sadoff, D. Pavliovka, R. Schneerson and J. B. Robbins, The Lancet, 1997, 349, 155-159). Conversion of polysaccharide T-independent antigens to T-dependent ones through their covalent attachment to a carrier protein has had a tremendous impact in the field of bacterial vaccines. Several such neoglycoconjugate vaccines are currently in use against Haemophilus influenzae b (R. W. Ellis and D. M. Granoff, Development and clinical use of Haemophilus b conjugate vaccines, Dekker, New York, 1994), Neisseria meningitidis (P. Richmond, R. Borrow, E. Miller, S. Clark, F. Sadler, A. Fox, N. Begg, R. Morris and K. Cartwright, J. Infect. Dir., 1999, 179, 1569-1572) and Streptococcus pneumoniae (M. B. Renels, K. M. Edwards, H. L. Keyserling, K. S. Reisinger, D. A. Hogerman, D. V. Madore, I. Chang, P. R. Paradiso, F. J. Malinoski and A. Kimura, Pediatrics, 1998, 101, 604-611). These polysaccharide-protein conjugate vaccines are highly complex structures, whose immunogenicity depends on several parameters amongst which are the length and nature of the saccharide component as well as its loading on the protein. It is reasonably admitted that control of these parameters is somewhat difficult when dealing with polysaccharides purified from bacterial cell cultures. As recent progress in carbohydrate synthesis allows access to complex saccharides, it has been suggested that the use of well-defined synthetic oligosaccharides may allow a better control, and consequently the optimisation, of these parameters. Indeed, available data on S. dysenteriae type 1 indicate that neoglycoconjugates incorporating di-, tri- or tetramers of the O—SP repeating unit were more immunogenic than a detoxified LPS-human serum albumin conjugate of reference (V. Pozsgay, C. Chu, L. Panell, J. Wolfe, J. B. Robbins and R. Schneerson, Proc. Natl. Acad. Sci. USA, 1999, 96, 5164-5197).
Besides, recent reports demonstrate that short oligosaccharides comprising one repeating unit may be immunogenic in animal models (B. Benaissa-Trouw, D. J. Lefeber, J. P. Kamerling, J. F. G. Vliegenthart, K. Kraaijeveld and H. Snippe, Infect. Immun., 2001, 69, 4698-4701; F. Mawas, J. Niggemann, C. Jones, M. J. Corbet, J. P. Kamerling and J. F. G. Vliegenthart, Infect. Immun., 2002, 70, 5107-5114). Another critical parameter in the design of neoglycoconjugate vaccines is the carrier protein. As potential applications for these vaccines are expanding, the need for new carrier proteins licensed for human use is growing (J. B. Robbins, R. Schneerson, S. C. Szu and V. Pozsgay in Polysaccharide-protein conjugate vaccines, vol. (S. Plotkin and B. Fantini Eds), Elsevier, Paris, 1996, pp. 135-143). That synthetic peptides representing immunodominant T-cell epitopes could act as carriers in polysaccharide and oligosaccharide conjugates has been suggested (G. J. P. H. Boons, P. Hoogerhout, J. T. Poolman, G. A. van der. Marel and J. H. van Boom, Bioorg. Med. Chem., 1991, 1, 303-308) and later on demonstrated (E. Lett, S. Gangloff, M. Zimmermann, D. Wachsmann and J.-P. Klein, Infect. Immun., 1994, 62, 785-792; A. Kandil, N. Chan, M. Klein and P. Chong, Glycoconjugate J., 1997, 14, 13-17). Besides, the use of T-cell epitopes offers several advantages, including potential access to well-defined conjugates with no risk of epitopic suppression, as this latter phenomenon appeared to be a major drawback of protein carriers (T. Barington, M. Skettrup, L. Juul and C. Heilmann, Infect. Immunol., 1993, 61, 432-438; M.-P. Schutze, C. Leclerc, M. Jolivet, F. Audibert and L. Chedid, J. Immunol., 1985, 135, 2319-2322). Polypeptides containing multiple T-cell epitopes have been generated in order to address the extensive polymorphism of HLA molecules (P. R. Paradiso, K. Dermody and S. Pillai, Vaccine Res., 1993, 2, 239-248). In other strategies, universal T-helper epitopes compatible with human use have been characterized, for example from tetanus toxoid (D. Valmori, A. Pessi, E. Bianchi and G. P. Corradin, J. Immunol., 1992, 149, 717-721), or engineered such as the pan HLA DR-binding epitope (PADRE) (J. Alexander, J. Sidney, S. Southwood, J. Ruppert, C. Oseroff, A. Maewal, K. Snoke, H. M. Serra, R. T. Kubo, A. Sette and H. M. Grey, Immunity, 1994, 1, 751-761). Recently, covalent attachment of the human milk oligosaccharide, lacto-N-fucopentose II, to PADRE resulted in a linear glycopeptide of comparable immunogenicity to that of a glycoconjugate employing human serum albumine (HAS) as the carrier (J. Alexander, A.-F. d. Guercio, A. Maewal, L. Qiao, J. Fikes, R. W. Chesnut, J. Paulson, D. R. Bundle, S. DeFrees and A. Sette, J. Immunol., 2000, 164, 1625-1633).
Based on these converging data, the inventors have focused on the development of well-defined neoglycoconjugate as an alternative to polysaccharide protein conjugate vaccines targeting infections caused by S. flexneri serotype 2a. The target neoglycoconjugates were constructed by covalently linking an immunocarrier, serving as T-helper epitope(s), to appropriate carbohydrate (oligo- or polysaccharide) haptens, serving as B epitopes mimicking the S. flexneri 2a O—Ag. To this end, a rationale approach involving a preliminary study of the interaction between the bacterial O—SP and homologous protective monoclonal antibodies, was employed to define the carbohydrate haptens.